DEGRADATION AND LOSS OF MATRIX PROTEINS FROM DEVELOPING ENAMEL
DEGRADATION AND LOSS OF MATRIX PROTEINS FROM DEVELOPING ENAMEL
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DOI：
10.1002/ar.1092240219
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发表时间：
1989-06-01
影响因子：
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通讯作者：
NANCI, A
中科院分区：
文献类型：
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作者：
SMITH, CE;POMPURA, JR;NANCI, A
利用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）、荧光照相术、放射自显影术和体外培养的方法，研究了发育中大鼠切牙釉质基质蛋白质分解和丢失的模式和时间。对于生物化学研究，罗宾逊等人的技术。(1974，1977，1983）用于将釉质器官和釉质沿牙齿长度沿着以1 mm间隔横切成一系列条带。提取每个条带中的蛋白质，并通过Lowry分析定量或施加到12%平板（釉质）或5 - 15%连续梯度（釉质器官）SDS-聚丙烯酰胺凝胶上，并通过电泳分离。生物化学研究表明，釉质条内所含的蛋白质量在整个分泌期逐渐增加，在成熟期早期达到高峰，然后迅速下降。在此期间，釉蛋白在SDS-聚丙烯酰胺凝胶上的分布发生了显着变化。这些变化包括某些分子量（如18 kDa）的蛋白质染色强度的增加和减少，以及在釉质形成分泌期开始时未清楚观察到的一些蛋白质（如32和10 kDa）的出现和消失。用35S-蛋氨酸进行的标记研究表明，形成釉质（分泌阶段）的典型蛋白质的"堆叠"排列实际上代表了蛋白质的非常动态的结合，新的蛋白质被添加到堆叠的顶部，然后随着时间的推移分解成较低分子量的蛋白质。在整个分泌阶段，新的蛋白质总是被添加到堆栈的顶部，但在早期成熟过程中，这种活动显着放缓，从而使老化蛋白质的分解更清晰。放射自显影研究与3H-甲硫氨酸表明，新分泌的蛋白质的分解也与标签的运动从网站的分泌到更深的，以前未标记的，形成釉质的地区。体外研究表明，釉质蛋白质的分解速度和程度有很大差异，这取决于提取蛋白质的釉质形成阶段。分泌期釉质蛋白在体外降解缓慢，积累的蛋白质接近18 kDa。早期成熟阶段的釉质蛋白质显示出更快速的分解，几乎没有积累的蛋白质接近18 kDa，而晚期成熟阶段的釉质蛋白质在体外孵育2天显示出完全降解。抑肽酶能有效抑制成熟期釉质蛋白的降解。这些结果支持的概念，釉原蛋白降解成小的多肽，通过细胞外蛋白酶的作用，其中至少有一个似乎是胰蛋白酶样丝氨酸蛋白酶。这些结果进一步表明，釉原蛋白开始在其分泌的数小时内分解。在釉质形成的分泌期和成熟期之间，参与釉质原蛋白降解的蛋白酶的活性和数量或类型似乎也有所增加。
The pattern and timing of the breakdown and loss of matrix proteins were studied in developing rat incisor enamel using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), fluorography, radioautography, and in vitro incubations of proteins isolated from freshly dissected, crushed pieces of enamel. For biochemical studies, the technique of Robinson et al.(1974, 1977, 1983) was used to transect the enamel organ and enamel into a series of strips at 1 mm intervals along the length of the tooth. The proteins in each strip were extracted and either quantified by Lowry analysis or applied to 12% slab (enamel) or 5-15% continuous gradient (enamel organ) SDS-polyacrylamide gels and separated by electrophoresis. The biochemical studies indicated that the amount of protein contained within an enamel strip increased gradually by volume across the secretory stage, reached a peak early during the maturation stage, and then declined rapidly thereafter. The distribution of enamel proteins on SDS-polyacrylamide gels changed markedly throughout this period. These changes included increases and decreases in the intensity of staining of proteins at certain molecular weights (eg, 18 kDa) and the appearance and disappearance of some proteins not seen clearly near the start of the secretory stage of amelogenesis (eg, 32 and 10 kDa). Labeling studies with 35S-methionine suggested that the “stacked” arrangement of proteins typical of forming enamel (secretory stage) actually represented a very dynamic association of proteins, with new ones being added at the top of the stack and then breaking down with time to become those seen at lower molecular weights. Across the secretory stage, new proteins were always added to the top of the stack, but during early maturation this activity slowed dramatically, allowing the breakdown of aging proteins to be visualized more clearly. Radioautographic studies with 3H-methionine indicated that the breakdown of newly secreted proteins also was correlated with a movement of label from the site of secretion into deeper, previously unlabeled, areas of forming enamel. In vitro studies revealed that the rate and degree of breakdown of enamel proteins varied markedly, depending on the stage of amelogenesis from which the proteins were extracted. Secretory stage enamel proteins showed slow in vitro degradation with accumulation of proteins near 18 kDa. Early maturation stage enamel proteins showed more rapid breakdown with little accumulation of proteins near 18 kDa, whereas late maturation stage enamel proteins showed complete degradation by 2 days of incubation in vitro. Degradation of maturation stage enamel proteins could be effectively inhibited with aprotinin. These results support the concept that amelogenins are degraded into small polypeptides through the action of extracellular proteinases, at least one of which appears to be a trypsinlike serine proteinase. These results further suggest that amelogenins begin to break down within hours of their secretion. There also appears to be an increase in the activity andlor amount or type (s) of proteinases involved in the degradation of amelogenins between the secretory and maturation stages of amelogenesis.